GLV-1h68 is an attenuated oncolytic recombinant vaccinia virus derived from LIVP strain. It was constructed by inserting three expression cassettes (encoding Renilla luciferase Aequorea green fluorescent protein fusion, beta-galactosidase, and beta-glucuronidase) into the F14.5L, J2R (encoding thymidine kinase) and A56R (encoding hemagglutinin) loci of the viral genome, respectively. GLV-1h68 is demonstrated the abilities to infect, replicate in, and lyse many human cancer cell lines both in vitro and in vivo. In this study, it was used a combination of 4 antibiotics to kill almost gut bacteria in mice bearing human lung cancer xenografts (A549), following by treatment with single dose of 5 x 10 exponent 6 PFU of GLV-1h68. The results showed that tumor sizes of antibiotic treated animals were smaller than the non-antibiotic treated animals. Concurrently, the number of viral particles in tumors of antibiotic treat for animals were found higher than the controls. This finding opens a new better option for cancer treatment by using combination of antibiotic and oncolytic therapies.